Having established in the previous project period, that mitotic activation is crucial for the neurodegenerative mechanism in AD, in this renewal application we will continue to 'walk up' the cell cycle signaling pathway in order to delineate upstream events leading to mitotic activation. We will use a combination of descriptive studies of human brain control to identify new players, and functional studies to elucidate their role in mitotic phosphoepitope formation. Thus the brain studies will generate a continuously evolving 'picture' of processes in AD, and the picture will be used to formulate hypotheses for the role of specific regulators, to be tested directly in our model systems. In Aim I we will continue to explore the mechanism of mitotic activation in neurons. II) We will complete identification of all known immediate upstream cdc2 regulators in brain, and test their roles in mitotic phosphoepitope formation in our cdc2/cyclin B neuronal line, using antisense and overexpression strategies. III) We will determine whether mitotic activation in AD is coupled to prior S phase events as in a normal cell cycle, by investigating the temporal and spatial relationship of S and M phase markers in AD brain and determining whether DNA replication occurs. IIII) We will determine whether the GI cdk4 kinase, a regulator of cell cycle entry, operates in a similar capacity in AD. The 'pictures' obtained from HI and 1111 will be further studied in an independent model of oncogenic ras expression in primary neurons in vitro and in transgenic mice in vivo. Unlike the cdc2/cyclinBl transgenic mouse that produces mitotic changes, the ras mouse will support complete progression of hierarchical cell cycle events leading to mitotic changes. In Aim II, we will determine whether DNA damage prevents mitosis in AD through DNA damage checkpoint activation. We will study the temporal and spatial relationship of DNA damage and cell cycle markers to form a picture that will be unraveled in functional studies with the ras model. In Aim III, we will examine the role of the cdc2/cyclin A complex in AD pathogenesis. We already have obtained a picture to suggest that this complex is important in AD. Here, using stable neurons with conditional cdc2/cyclin A expression, we will determine whether the complex generates mitotic or other AD phosphoepitopes, and potentiates the effects of cdc2/cyclin Bi. The pursuit of the above three aims will help connect the spurious appearances of various cell cycle elements in AD, and attempt to solidify their involvement in a cell cycle driven mechanism of neurodegeneration. This work will lead to the use of cell cycle inhibitors.